Combining atomic-resolution structure with high-resolution tracking in cells to dissect regulation and mechanism of the MKlp2 kinesin

• Applicant: Professor Dr. Jörg Enderlein, since 6/2019
• Subject Area: Biophysics ; Structural Biology
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 283958999

Mitosis involves a complex coordination of cytoskeletal rearrangements for which molecular motors are temporally and spatially controlled to precisely divide the cell and distribute the chromosomes. The most burning open questions in this area concern the collective regulation of motors, which is also central to drug development. The kinesin 6 MKlp2 plays critical roles for the metaphase to anaphase transition and for cytokinesis. Inhibition of MKlp2 function in pancreatic adenocarcinoma cells reduced cell growth and MKlp2 inhibitors can kill tumor stem cells. The MKlp2 motor domain is 60% larger than that of other kinesins, due to several unique inserts that are believed to be involved in the regulation of this kinesin. MKlp2 interacts with the kinases polo-kinase 1 (Plk1) and AuroraB and controls their spatial and temporal action during cell division. MKlp2 also interacts with myosin II which is critical to bring kinases to the proper place for furrow ingression. Whether the motor activity of Mklp2 is important at this location and whether Myosin II activity is influenced by MKlp2 is unknown. The motor properties of MKlp2 and its precise cellular functions, as well as its regulation remain elusive. MKlp2 is a N-terminal kinesin with unique features, including an N-terminal extension, a short insertion in loop2 (important for MT binding), a long insertion in loop6 (L6) adjacent to the N-terminal extension in the tertiary structure, and a neck linker that is about four times longer than that of other kinesins. Because the large insertions are near the structural elements important for force generation, this motor is likely to have a different mechanism for generating its powerstroke. Whether the insertions contribute to and/or regulate its role in cytokinesis is unclear. Our preliminary data on how the motor interacts with microtubules confirms that this kinesin is unusual. The interaction with Plk1 located close to the mechanical element of this kinesin is likely to modulate how this motor produces force and how such force is used to allow transport of cargos or to organize microtubules during mitosis. To understand how MKlp2 functions, we propose to combine high-resolution structure determination with a functional characterization in vitro and in vivo. For single-molecule studies in vivo we will use a novel approach using single-walled carbon nanotubes (SWNTs) as precisely targetable and uniquely stable near-infrared fluorescent markers. We further have established multidisciplinary international collaborations to add transient kinetics and cryo-electron microscopy (cryoEM) experiments to study basic properties of this motor. The project is ambitious and innovative by combining atomic structure determination with functional single-molecule studies in live cells to define the properties of the motor and we will break new ground by tracking the motors in live cells using novel super-resolution live-cell imaging technology.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Co-Investigator: Professorin Dr. Anne Houdusse, Ph.D.
• Ehemaliger Antragsteller: Professor Dr. Christoph Friedrich Schmidt, until 5/2019